Slow release protein polymers

ABSTRACT

The invention features articles for delivery of a biologically active substance, methods for making such articles, and methods for treating an animal using the articles.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of and claims priority from U.S. Ser.No. 09/772,174, filed Jan. 29, 2001 now U.S. Pat. No. 6,699,504; whichclaims benefit of U.S. Ser. No. 60/178,852, filed Jan. 28, 2000,entitled “Slow Release Protein Polymers,” having as inventors Steven C.Rowe, Kalvin Yim, Beadle P. Retnarajan, and Jeffrey A. Hubbell.

BACKGROUND OF THE INVENTION

The invention relates to biodegradable compositions forsustained-release drug delivery and methods for administering abiologically active substance via these compositions.

Rapid advances in the fields of genetic engineering and biotechnologyhave led to the development of an increasing number of proteins andpolypeptides that are useful as pharmaceutical agents. The developmentof methods for administering these new pharmaceutical agents is thusbecoming increasingly important.

Most proteins have relatively short half-lives, requiring frequentadministration to achieve efficacious blood levels. To increase patientconvenience and to improve efficacy and safety by keeping blood levelswithin the therapeutic range, smoothly releasing injectable depotformulations of protein drugs are highly desirable.

Recent polymer developments have improved the ability to deliverproteins and peptides by allowing for slower and steadier release of themolecule in the patient's system. However, in many cases, the activeform of the protein is difficult to formulate in biodegradable polymers.Synthetic materials, such as biodegradable hydrogels, have also beendeveloped for use in delivering proteins. Despite the advances providedby the available polymers and hydrogels, the delivery of protein to thesystemic and local circulation is still relatively rapid, in some casestoo rapid to allow this route of administration to be used.

SUMMARY OF THE INVENTION

The present invention features articles for delivery of a biologicallyactive substance (hereafter “BAS”), and methods for making sucharticles. The articles of the invention improve the bioavailability ofthe BAS by formulating the BAS in an insoluble form. The invention alsofeatures methods of treating an animal using the articles for deliveryof a BAS.

Accordingly, in a first aspect the invention features a biocompatibletherapeutic article for delivery of a BAS, comprising a macromer, amolecule or mixture of molecules which preferentially excludes proteins,and the BAS, wherein the BAS is in an insoluble format upon completionof the formulation of the article comprising the macromer, molecule, ormixture of molecules which preferentially excludes proteins, and BAS.

In a preferred embodiment of the first aspect of the invention, thebiocompatible therapeutic article has at least one of the followingproperties: the BAS is less than 15% aggregated; the article contains atleast 10% macromer and at least 5% BAS, as measured by dry weight; thetime at which 5% of the releasable BAS is released from the article isgreater than 1/16 of t₅₀; or the t₅₀ is greater than or equal to ⅝ oft₈₀. More preferably the biocompatible therapeutic article has at leasttwo of the above properties. Most preferably, the biocompatibletherapeutic article has all of the above properties.

In another embodiment of the first aspect of the invention, the moleculewhich preferentially excludes proteins is a macromer, poly(ethyleneglycol), hyaluronic acid, or poly(vinylpyrrolidone). In yet anotherembodiment, the macromer is a hydrogel. In still another embodiment, thesolubility of a protein in the article comprising the macromer, moleculethat preferentially excludes proteins, and BAS is less than 5-10 mg/ml,and more preferably is less than 1 mg/ml.

In another embodiment of the first aspect of the invention, the mixtureof molecules comprises a positively charged ion-carrying reagent, forexample, triethanolamine or Tris, when the pH is such that the proteinis negatively charged. In still another embodiment, the mixture ofmolecules comprises a negatively charged ion-carrying reagent, such assodium dodecyl sulfate, when the pH is such that the protein ispositively charged. In yet another embodiment, the mixture of moleculescomprises a surfactant, for example, Tween 20, Tween 80, or poloxamerF68. In a second aspect, the invention features a method for making atherapeutic article for delivery of a BAS, involving (a) combining theBAS with a molecule or mixture of molecules which preferentiallyexcludes proteins; (b) combining the mixture formed in step (a) with amacromer, wherein the BAS is in an insoluble form and remains insolubleupon combining with the molecule or mixture of molecules whichpreferentially excludes proteins and the macromer; (c) forming a mixtureof the combination formed in step (b); and (d) polymerizing the mixtureto form an article.

In one embodiment of the second aspect of the invention, steps (a) and(b) are combined into a single combination step.

In a preferred embodiment of the second aspect of the invention, thebiocompatible therapeutic article has at least one of the followingproperties: the BAS is less than 15% aggregated; the article contains atleast 10% macromer and at least 5% BAS, as measured by dry weight; thetime at which 5% of the releasable BAS is released from the article isgreater than 1/16 of t₅₀; or the t₅₀ is greater than or equal to ⅝ oft₈₀. More preferably the biocompatible therapeutic article has at leasttwo of the above properties. Most preferably, the biocompatibletherapeutic article has all of the above properties.

In another embodiment of the second aspect of the invention, themolecule which preferentially excludes proteins is a macromer,poly(ethylene glycol), hyaluronic acid, or poly(vinylpyrrolidone). Inyet another embodiment, the macromer is a hydrogel. In yet anotherembodiment, the macromer is a hydrogel. In still another embodiment, thesolubility of a protein in the article comprising the macromer, moleculethat preferentially excludes proteins, and BAS is less than 5-10 mg/ml,and more preferably is less than 1 mg/ml.

In another embodiment of the second aspect of the invention, the mixtureof molecules comprises a positively charged ion-carrying reagent, forexample, triethanolamine, when the pH is such that the protein isnegatively charged. In still another embodiment, the mixture ofmolecules comprises a negatively charged ion-carrying reagent, such assodium dodecyl sulfate, when the pH is such that the protein ispositively charged. In yet another embodiment, the mixture comprises asurfactant, for example, Tween 20, Tween 80, or poloxamer F68.

In a third aspect the invention features a method of treating an animal,involving administering the biocompatible therapeutic article of thefirst aspect of the invention to a mammal. Preferably the mammal is arodent, and most preferably the mammal is a human.

In yet other preferred embodiments, the articles are administered to thelung of the mammal, or are administered intravenously, subcutaneously,intramuscularly, orally, or nasally.

In a preferred embodiment of any of the above aspects of the invention,the macromer comprises: (a) a region forming a central core; (b) atleast two degradable regions attached to the core; and (c) at least twopolymerizable end groups, where the polymerizable end groups areattached to the degradable regions. In preferred embodiments, the regionforming a central core is a water soluble region. The water solubleregion may be poly(ethylene glycol), poly(ethylene oxide), poly(vinylalcohol), poly(vinylpyrrolidone), poly(ethyloxazoline), poly(ethyleneoxide)-co-poly(propylene oxide) block copolymers, polysaccharides,carbohydrates, proteins, and combinations thereof. The degradable regionis selected from the group consisting of poly(α-hydroxy acids),poly(lactones), poly(amino acids), poly(anhydrides), poly(orthoesters),poly(orthocarbonates), and poly(phosphoesters). Preferably, thepoly(α-hydroxy acid) is poly(glycolic acid), poly(DL-lactic acid), orpoly(L-lactic acid), and the poly(lactone) is poly(ε-caprolactone),poly(δ-valerolactone), or poly(γ-butyrolactone). In another preferredembodiment, the degradable region comprises poly(caprolactone). In yetanother embodiment, the polymerizable end groups contain a carbon-carbondouble bond capable of polymerizing the macromer.

In other embodiments of the above aspects of the invention, the macromerincludes: (a) a water soluble region comprising a three-armedpoly(ethylene glycol) with a molecular weight of 3,000 to 6,000 daltons;(b) lactate groups attached to the region in (a); and (c) acrylategroups capping the region in (b). The macromer may alternativelyinclude: (a) a water soluble region comprising poly(ethylene glycol)with a molecular weight of either 2,000 or 3,400 daltons; (b) lactategroups on either side of the region in (a); and (c) acrylate groupscapping either side of the region in (b). In another alternative, themacromer may include (a) a water soluble region comprising poly(ethyleneglycol) with a molecular weight of 3,400 daltons; (b) caprolactonegroups on either side of region in (a); and (c) acrylate groups cappingeither side of the region in (b).

In still other embodiments of any of the above aspects of the invention,the article includes at least 5%, more preferably 10%, and mostpreferably 20-30% active substance by dry weight. In still anotherembodiment, the article is biodegradable.

In a more preferred embodiment of any of the above aspects of theinvention, the macromer includes a water soluble region consisting of athree-armed PEG with a molecular weight of 4,200 to 5,400 daltons;lactate groups one end of each arm of the PEG; and acrylate groupscapping the lactate groups.

In another more preferred embodiment of the above aspects of theinvention, the macromer is made of a triad ABA block copolymer ofacrylate-poly(lactic acid)-PEG-acrylate-poly(lactic acid)-acrylate. ThePEG has a MW of 3,400 daltons; the poly(lactic acids) on both sides hadan average of about five lactate units per side; and the macromer istherefore referred to herein as “3.4kL5.” In another more preferredembodiment, a lower molecular weight PEG, such as MW 2,000 daltons PEGis used in place of the MW 3,400 PEG, and the resulting macromer isabbreviated as “2kL5.”

In yet another more preferred embodiment of the above aspects of theninvention, the macromer is an acrylate-PCL-PEG-PCL-acrylate macromer.The PEG has a MW of 3,400 daltons and has polycaprolactone on bothsides, with an average of about 6 caproyl units per side. This macromeris referred to herein as “3.4kC6.”

In other preferred embodiments, the BAS is a protein or peptide. Morepreferably the protein is chosen from a group consisting of hormones,antibodies, differentiation factors, angiogenic factors, enzymes,cytokines, chemokines, interferons, colony-stimulating factors, andgrowth factors. Most preferably, the protein is a hormone, such as humangrowth hormone, or a peptide, such as LHRH.

In still other embodiments of the second and third aspects of theinvention, the therapeutic articles release at least 80% of the BAS at atime 1¼ times greater than t₅₀. At least 80% of the therapeutic articlesmay have a particle size of less than about 80 microns. The watersoluble region may consist essentially of PEG having a molecular weightof about 500 to 20,000 daltons, and more preferably, between 1,000 and10,000 daltons. The degradable region may comprise a blend of at leasttwo different polymers. In addition, the macromer may be non-degradable.

In still other embodiments of the second and third aspects of theinvention, the therapeutic article is capable of releasing the BAS forat for a period of time at least 2 times greater than t₅₀. The articleis also capable of delivering a therapeutic dose of the BAS for at for aperiod of time at least 1¼ times greater than t₅₀.

By “macromer” is meant a polymer with three components: (1) abiocompatible, water soluble region; (2) a biodegradable/hydrolyzableregion, and (3) at least two polymerizable regions.

By “biologically active substance” or “BAS” is meant a compound, be itnaturally-occurring or artificially-derived, that is incorporated intoan article and which may be released and delivered to a site.Biologically active substances may include, for example, peptides,polypeptide, proteins, synthetic organic molecules, naturally occurringorganic molecules, nucleic acid molecules, and components thereof.

By “a molecule or mixture of molecules that preferentially excludesproteins” is meant a molecule or mixture of molecules, be itnaturally-occurring or artificially-derived, that, when added to asolution, confers a lower level of solubility of the protein orpolypeptide in said solution. Preferably, protein solubility will bedecreased 50-fold; more preferably, 100-fold and most preferably about200-fold. Preferably the solubility of a protein in a solution thatincludes said molecule or mixture of molecules that preferentiallyexcludes proteins is less than 5-10 mg/ml, and more preferably is lessthan 1 mg/ml.

By “substantially pure polypeptide” or “protein” s meant a polypeptideor protein that has been separated from the components that naturallyaccompany it. The terms polypeptide and protein may be usedinterchangeably. Typically, the polypeptide is substantially pure whenit is at least 60%, by weight, free from the proteins andnaturally-occurring organic molecules with which it is naturallyassociated. A substantially pure polypeptide may be obtained, forexample, by extraction from a natural source (e.g., a cell expressingthe desired polypeptide), by expression of a recombinant nucleic acidencoding a desired polypeptide, or by chemically synthesizing thepolypeptide. Purity can be assayed by any appropriate method, e.g., bycolumn chromatography, polyacrylamide gel electrophoresis, agarose gelelectrophoresis, optical density, or HPLC analysis.

A protein is substantially free of naturally associated components whenit is separated from those contaminants which accompany it in itsnatural state.

Thus, a protein which is chemically synthesized or produced in acellular system different from the cell from which it naturallyoriginates will be substantially free from its naturally associatedcomponents. Accordingly, substantially pure polypeptides include thosederived from eukaryotic organisms but synthesized in E. Coli or otherprokaryotes.

By “purified nucleic acid” is meant a nucleic acid that is free of thegenes which, in the naturally-occurring genome of the organism fromwhich the nucleic acid of the invention is derived, flank the gene. Theterm therefore includes, for example, a recombinant DNA which isincorporated into a vector; into an autonomously replicating plasmid orvirus; or into the genomic DNA of a prokaryote or eukaryote; or whichexists as a separate molecule (e.g., a cDNA or a genomic or cDNAfragment produced by PCR or restriction endonuclease digestion)independent of other sequences. It also includes recombinant DNA whichis part of a hybrid gene encoding additional polypeptide sequence.

By “biocompatible” is meant that any compound or substance which isadministered to a subject, cell, or tissue is used to treat, replace, oraugment a function of the subject, cell or tissue, and is not harmful tosaid function.

By “insoluble” is meant that the solubility of a compound is less than 1g/100 ml in a solution. The solution may be an aqueous solution, anorganic solvent, such as dimethylsulfoxide, or a mixture of aqueous andorganic solvents. As used herein, a BAS is in an insoluble format uponcompletion of the formulation for a therapeutic article for delivery ofthe BAS. The BAS remains in an insoluble format upon delivery of thetherapeutic article to a patient, and is then slowly released at acontrolled rate for localized or systemic delivery to the patient. Asused herein, by “aggregated” is meant that a BAS is releasable asindividual molecules. The percent of a BAS in an article which isaggregated can be determined, for example, by SEC-HPLC.

By “therapeutic dose,” when referring to a BAS, is meant a plasma levelbetween the minimum effective level and the toxic level.

By a “mixture” is meant a composition in which all of the compoundscontained in the composition are evenly distributed.

As used herein, by “pore size” is meant the dimensions of a space in theintact polymer through which a macromer, component of a macromer, or aBAS potentially can pass. Pore sizes which are utilized as part of theinvention are those smaller than the BAS as it is present in theparticular embodiment (e.g., a protein molecule, or aggregate thereof).

As used herein, by “period of release” is meant the length of time ittakes for a specified percent of the BAS to be released from an article.The period of release may be assessed, for example, by measuring thetime it takes for 50% or 80% of the BAS to be released from the article.

By “low burst effect” is meant that the amount of BAS released from anarticle is released relatively steadily over time, rather than at aninitial fast rate, followed by a slower rate. For example, a BAS has alow burst effect (e.g., less than or equal to 20% burst) upon releasefrom an article when the period of release for 5% of the releasable BASis greater than 1/16 of t₅₀, or when the t₅₀ is greater than or equal to⅝ of t₈₀. In contrast to a low burst article, a high burst article(e.g., one which rapidly releases 30% of the BAS) might release 5% ofits releasable BAS in less than 1/18 of t₅₀ and have a t₅₀ equal to 1/2of t₈₀.

A specific example of a low burst product of the present invention isone in which less than 20% of the BAS comes out in the first day for aproduct designed to release a BAS for 10 days.

By “t₅₀” is meant the time at which 50% of the original load of BAS hasbeen released. As used herein, preferably 5% of the releasable BAS isreleased at a time which is greater than 1/16 of t₅₀, or the t₅₀ isgreater than or equal to ⅝ of the t₈₀.

By “t₈₀” is meant the time at which 80% of the original load of BAS hasbeen released. As used herein, preferably 5% of the releasable BAS isreleased at a time which is greater than 1/16 of t₅₀, or the t₅₀ isgreater than or equal to ⅝ of the t₈₀.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is the release profile of microspheres made with precipitatedhGH.

FIG. 2 is the release profile of hGH from 2kL5 microspheres.

FIG. 3 is a graph depicting the release of spray-dried bSA from4.2kL3-A3 microspheres.

FIG. 4 is a graph of the effects of biologically active particle size onthe release of bovine serum albumen (bSA) from 30% 3.4kL5.

FIG. 5 is a graph of the effect of biologically active particle size andprotein loading on the release of fine-ground bSA (10% loaded) andvarious crystalline particles (1-10% loaded) from 3.4kL5.

FIG. 6 is a graph of the effects of biologically active particle size onthe release of human growth hormone (hGH) from 30% 3.4kL5.

FIG. 7 is a graph of the effect of microsphere pore size on release ofmicronized hGH from articles.

DETAILED DESCRIPTION

The invention provides methods and compositions for the administrationof a biologically active substance (BAS) in an insoluble format. Thecompositions of the invention improve the bioavailability of the BAS byformulating the BAS in an insoluble format. These methods andcompositions provide for the controlled, sustained delivery ofrelatively large quantities of these substances, with a low bursteffect.

Macromers

The macromers of the present invention have at least one region forminga central core, at least one degradable (e.g., hydrolyzable) region, andat least one polymerizable region. The macromers may be water-soluble orwater insoluble. Preferably, the region forming a central core is watersoluble. If desired, the macromers may be polymerized to form hydrogels,which are useful for delivering incorporated substances at a controlledrate. Methods to formulate macromers and shape them into articles aredescribed, for example in WO 99/03454, hereby incorporated by reference.An important aspect of the macromers is that the polymerizable regionsare separated by at least one degradable region. This separationfacilitates uniform degradation in vivo.

The ratio between the central core region and the hydrolyzable region ofthe macromer determines many of the general properties of the macromer.For example, the water solubility of the macromers can be controlled byvarying the percentage of the macromer that consists of hydrophobicdegradable groups.

There are several variations of the macromers of the present invention.For example, the polymerizable regions can be attached directly to thedegradable regions; alternatively, they can be attached indirectly viawater-soluble, nondegradable regions, with the polymerizable regionsseparated by a degradable region. For example, if the macromer containsa single water-soluble region coupled to a degradable region, onepolymerizable region can be attached to the water-soluble region, andthe other to the degradable region.

In another embodiment, a water-soluble region forms the central core ofthe macromer and has at least two degradable regions attached to it. Atleast two polymerizable regions are attached to the degradable regionsso that, upon degradation, the polymerizable regions, particularly inthe polymerized gel form, are separated. Alternatively, if the centralcore of the macromer is formed by a degradable region, at least twowater soluble regions can be attached to the core, and polymerizableregions are attached to each water soluble region.

In still another embodiment, the macromer has a water-soluble backboneregion, with a degradable region attached to the macromer backbone. Atleast two polymerizable regions are attached to the degradable regions,such that they are separated upon degradation, resulting in gel productdissolution. In a further embodiment, the macromer backbone region isformed of a degradable backbone region having water-soluble regions asbranches or grafts attached to the degradable backbone. Two or morepolymerizable regions are attached to the water soluble branches orgrafts.

In another variation, the macromer backbone may have multiple arms;e.g., it may be star-shaped or comb-shaped. The backbone may include awater-soluble region, a biodegradable region, or a water-soluble,biodegradable region. The polymerizable regions are attached to thisbackbone. Again, the polymerizable regions must be separated at somepoint by a degradable region.

Throughout the specification, the following abbreviations are sometimesused to describe the specific macromers of the invention. In threeparticular examples, a macromer having a water soluble region consistingof PEG with a molecular weight of 4,000 daltons, with 5 lactate groupson either side of this region, capped on either side with acrylategroups, is referred to as “4kL5.” Similarly, a macromer having a watersoluble region consisting of PEG with a molecular weight of 3,400daltons, with 6 caprolactone groups on either side of this region,capped on either side with acrylate groups, is referred to as “3.4kC6.”Likewise, a macromer having a water soluble region consisting of PEGhaving a molecular weight of 5,400 daltons and 3 arms, each armcontaining 3 lactate groups, extending from this region, capped oneither side with acrylate groups, is referred to as “4.2kL3-A3.”

Water-Soluble Region

In preferred embodiments, the central core is a water soluble region.This water soluble region of the macromer may include poly(ethyleneglycol), poly(ethylene oxide), poly(vinyl alcohol),poly(vinylpyrrolidone), poly(ethyloxazoline), poly(ethyleneoxide)-co-poly(propylene oxide) block copolymers, polysaccharides,carbohydrates, or proteins, or combinations thereof.

The macromer preferably comprises a water soluble core region comprisingPEG, as PEG has high hydrophilicity and water solubility, as well asgood biocompatibility. The PEG region preferably has a molecular weightof about 400 to about 40,000 daltons, and more preferably has amolecular weight of about 1,000 to about 30,000 daltons, about 1,000 toabout 20,000 daltons, or about 2,000 to about 10,000 daltons.

Degradable Region

The degradable region of the macromer may contain, for example,poly(α-hydroxy acids), poly(lactones), poly(amino acids),poly(anhydrides), poly(orthoesters), poly(orthocarbonates) orpoly(phosphoesters), or blends or copolymers of these polymers.

Exemplary poly(α-hydroxy acids) include poly(glycolic acid),poly(DL-lactic acid), and poly(L-lactic acid). Exemplary poly(lactones)include poly(ε-caprolactone), poly(δ-valerolactone),poly(γ-butyrolactone), poly(1,5-dioxepan-2-one), and poly(trimethylenecarbonate).

The degradable region may comprise a blend of at least two differentpolymers. Examples of copolymers include a copolymer of caprolactone andglycolic acid; and a copolymer of caprolactone and lactic acid.

Polymerizable Region

The polymerizable regions of the macromer preferably containcarbon-carbon double bonds capable of polymerizing the macromers. Thechoice of an appropriate polymerizable group permits rapidpolymerization and gelation. Polymerizable regions containing acrylatesare preferred because they can be polymerized using several initiatingsystems, as discussed below. Examples of acrylates include acrylate,methacrylate, and methyl methacrylate.

Polymerization of Macromers

If desired, the macromers of the present invention may be polymerizedusing polymerization initiators under the influence of long wavelengthultraviolet light, visible light, thermal energy, or a redox system. Thepolymerization can be conducted at room temperature or at lowertemperatures, for example, temperatures less than 20° C. Duringpolymerization, substances such as proteins are physically incorporatedinto the resulting polymer network of the hydrogel.

Polymerization of the macromers may be initiated in situ by light havinga wavelength of 320 nm or longer. When the polymerizable region containsacrylate groups, the initiator may be any of a number of suitable dyes,such as xanthine dyes, acridine dyes, thiazine dyes, phenazine dyes,camphorquinone dyes, acetophenone dyes, or eosin dyes withtriethanolamine, 2,2-dimethyl-2-phenyl acetophenone, and2-methoxy-2-phenyl acetophenone.

The polymerization may also take place in the absence of light. Forexample, the polymerization can be initiated with a redox system, usingtechniques known to those of skill in the art. In some cases it isadvantageous to polymerize macromers using the redox system of theinvention, as radical initiator production occurs at reasonable ratesover a wide range of temperatures.

Initiators that can be used in the redox system include, withoutlimitation, peroxides such as acetyl, benzoyl, cumyl and t-butyl;hydroperoxides such as t-butyl and cumyl, peresters such as t-butylperbenzoate; acyl alkylsulfonyl peroxides, dialkyl peroxydicarbonates,diperoxyketals, ketone peroxide, azo compounds such as2,2′-azo(bis)isobutyronitrile (AIBN), disulfides, and tetrazenes.

Shaping of Articles

The articles of the present invention may be formed in any shapedesired. For example, the articles may be shaped to fit into a specificbody cavity. They may also be formed into thin, flat disks or particles,such as microspheres. Alternatively, the articles may be shaped, thenprocessed into the desired shape before use, or ground into fineparticles. The desired shape of the article will depend on the specificapplication.

Macromer particles may be prepared using techniques known in the art,including single and double emulsion solvent evaporation, spray drying,and solvent extraction. As used herein, the term “particles” includes,but is not limited to, microspheres. In a microsphere, a BAS isdispersed throughout the particle. The particles may have a smooth orirregular surface, and may be solid or porous. Methods for makingmicrospheres are described in the literature, for example, in U.S. Pat.No. 4,272,398, Mathiowitz and Langer (J. Controlled Release 5:13-22(1987)); Mathiowitz et al. (Reactive Polymers 6:275-283 (1987));Mathiowitz et al. (J. Appl. Polymer Sci. 35:755-774 (1988)); Mathiowitzet al. (Scanning Microscopy 4:329-340 (1990)); Mathiowitz et al. (J.Appl. Polymer Sci., 45:125-134 (1992)); and Benita et al. (J. Pharm.Sci. 73:1721-1724 (1984)), hereby incorporated by reference. In onepreferred embodiment of the present invention, the microspheres areformed into hydrogel droplets.

In solvent evaporation, described, for example, in Mathiowitz, et al.,(1990), Benita et al. (1984), and U.S. Pat. No. 4,272,398, a polymer isdissolved in a volatile organic solvent, such as methylene chloride. Anagent to be incorporated, either in soluble form or dispersed as fineparticles, is optionally added to the polymer solution, and the mixtureis suspended in an aqueous phase that contains a surface active agentsuch as poly(vinyl alcohol). The resulting emulsion is stirred untilmost of the organic solvent evaporates, leaving solid microspheres,which may be washed with water and dried overnight in a lyophilizer.

In solvent removal, as described, for example, by Park et al. (J.Controlled Release 55:181-191 (1998)), a therapeutic or diagnostic agentis dispersed or dissolved in a solution of a selected polymer in avolatile organic solvent such as methylene chloride. The mixture canthen be suspended in oil, such as silicon oil, by stirring, to form anemulsion. As the solvent diffuses into the oil phase, the emulsiondroplets harden into solid polymer microspheres.

Processes for preparing ultrafine particles of biological molecules byatomizing liquid solutions of the macromolecules, drying the dropletsformed in the atomization step, and collecting the particles aredescribed in PCT WO 97/41833, hereby incorporated by reference.

Spray drying is implemented by passing a homogenous mixture of a BAS,such as a therapeutic agent, and the polymerizable macromer used to forma hydrogel through a nozzle, spinning disk, or equivalent device toatomize the mixture to form fine droplets. The substance and thepolymerizable macromer may be provided in a solution or suspension, suchas an aqueous solution. The fine droplets are exposed to light to causepolymerization of the macromer and formation of the hydrogel dropletsincorporating the substance. Hydrogels may be formed according to themethods described in U.S. Pat. No. 5,410,016, hereby incorporated byreference, or other techniques known in the art of polymer chemistry.

In another embodiment, hydrogel particles are prepared by a water-in-oilemulsion process, wherein the polymerizable macromers and the substanceto be incorporated are suspended in a water-in-oil emulsion and exposedto light to polymerize the macromers to form hydrogel particlesincorporating the substance, such as a BAS. Typically, polymerizationmay be conducted at room temperature.

The microspheres prepared using the techniques described above arefreeze dried, so they have a long shelf life (without biodegradation)and the BAS remains biologically active. Prior to use for injectableformulations, the microspheres are reconstituted in a suitable solution,such as saline or other liquids. For pulmonary delivery, either freezedried or reconstituted particles may be used.

Properties of the Macromers

The articles of the present invention are biodegradable. Biodegradationoccurs at the linkages within the extension oligomers and results infragments which are non-toxic and easily removed from the body and/orare normal, safe chemical intermediates in the body. These materials areparticularly useful for the delivery of hydrophilic materials, since thewater soluble regions of the polymer allow water to access the materialstrapped within the polymer.

Use of the Macromers

Macromers can be shaped into articles, for example, microspheres, andthese articles are capable of degrading under in vivo conditions atrates which permit the controlled release of incorporated substances.Release of such a substance may occur by diffusion of the substance fromthe polymer prior to degradation and/or by diffusion of the materialfrom the polymer as it degrades. Degradation of the polymer facilitateseventual controlled release of free macromolecules in vivo by gradualhydrolysis of the terminal ester linkages. The burst effects that aresometimes associated with other release systems are thus avoided in arange of formulations.

The rate of release of a BAS depends on many factors, for example, thecomposition of the water soluble region, the degree of polymerization ofthe macromer. The rate of release of a BAS also depends on the rate ofdegradation of the degradable region of the macromer. For example,glycolic esters lead to very rapid degradation, lactic esters tosomewhat slower degradation, and caprolactic esters to very slowdegradation. When the degradable region consists of polyglycolic acid,the release period is less than one week. When the degradable regionconsists of poly(lactic acid), the release period is about one week.When the degradable region consists of a copolymer of caprolactone andlactic acid or a copolymer of trimethylene carbonate and lactic acid,the release period is two to four weeks. When the degradable regionconsists of poly(trimethylene carbonate) or a copolymer of caprolactoneand trimethylene carbonate, the release period is about three to eightweeks. When the degradable region consists of poly(trimethylenecarbonate) or poly(caprolactone), the release period is longer thanabout five weeks.

The precise rate of release of a BAS from an article can be furthermodified by altering the ratio of hydrophilic and hydrophobic componentsof the article. For example, a very soluble macromer will yield, afterpolymerization, a hydrophilic gel; hydrophilic hydrogels have been shownto degrade more rapidly than hydrophobic ones. A blend of a hydrophilicmacromer (e.g., 4kL5) with a hydrophobic water insoluble macromer(3.4kC6) is used to form a polymerized hydrogel. This hydrogel will havea release rate that is in between the release rate of a hydrogelcontaining only lactic acid and a hydrogel containing only caprolactone.A macromer in which the degradable region is a copolymer of caprolactoneand lactic acid will also have a release rate which is in between therelease rate of a hydrogel containing only lactic acid and a hydrogelcontaining only caprolactone as the primary degradable group. Similarly,hydrophilicity of the active substance also affect the release rate ofthe BAS, with hydrophilic active substances generally released fasterthan hydrophobic substances.

The rate of release of a given BAS from a therapeutic article depends onthe quantity of the loaded substance, as a percent of the final productformulation. For example, it is generally thought in the polymer fieldthat while a large amount of BAS loading results in a longer period oftherapeutic dose delivery, it also results in a large burst effect.Therefore, an article which is loaded with a high amount of a BAS, andwhich also exhibits a low burst effect would be an optimal article. Thearticles or the present invention exhibit these characteristics.

Other factors which affect the release rate of a BAS from an article arethe aggregation and the solubility of the BAS. In order for the articlesof the present invention to have release profiles which are optimal fordelivering a BAS, the percent of the BAS which is aggregated should below. The articles of the present invention contain BAS which arepreferably less than 15% aggregated. In preferred embodiments, thearticles have this characterization of low aggregation even when theyand contain at least 2.5% BAS by dry weight, more preferably at least5%, and most preferably 20 or 40% by dry weight.

As stated above, another factor which affects the rate of release of aBAS from an article is the solubility of the BAS in the article. In thefield of polymer chemistry, it has generally been thought thatwater-soluble substances, such as a BAS, will yield homogenous systemswhen incorporated into the macromers of the invention. It has also beenthought that substances that do not solubilize in water within the timeit takes to form the macromers of the invention will yield heterogenoussystems. While the amount of burst in the heterogenous systems can beminimized by using a particulate suspension with small particles, it isgenerally thought that substances should be in a water soluble formatfor optimal delivery in a polymer delivery system. The articles of thepresent invention contain a BAS in an insoluble format, and thesearticles exhibit a low burst effect, an unexpected result.

Yet another factor that affects the release rate of a BAS from anarticle is the particle size of the BAS. For example, the articles ofthe present invention feature a BAS which has been ground and sieved toisolate fine particles which are smaller than approximately 75 micronsin any dimension. These particles were used to generate microspheres andthe release of the BAS from the microspheres was measured. This releaserate was compared to the release rate of the same BAS from the samemicrospheres, with the exception that the BAS was not fine-ground. Theresults of these studies indicated that a BAS which is fine-groundresults in release rates which are slower and have a low burst effect.By adjusting the factors discussed above, degradation and controlledrelease may be varied over very wide ranges. For example, release may bedesigned to occur over hours, days, or months.

The methods of the invention can produce particles that behave ashomogenous drug delivery systems. Because of the homogenous nature ofthe articles of the invention, there is no initial burst of releasedsubstance. In addition, the uniform consistency makes it possible toincorporate relatively high amounts of protein, while still minimizingthe burst effect.

The present invention also features insoluble macromers. These macromerscontain at least one water-soluble region, at least one degradable(e.g., hydrolyzable) region, and at least one polymerizable region. Thedegradable region contains polymers of glycolic acid, lactic acid,caprolactone, trimethylene carbonate, or blends or copolymers thereof.The degradable region must be water insoluble. For example, a macromerhaving a degradable region containing 15-20 lactide units can beprepared; this macromer will provide a relatively fast release rate. Amacromer with a degradable region containing 6 caprolactone units willprovide a relatively slow release rate. A macromer with a degradableregion containing a copolymer of 6 caprolactone units, 4 lactide units,and 4 glycolide units will provide a fast release rate, and a macromerwith a degradable region containing a copolymer of 3 lactide units and 7trimethylene carbonate units will provide an intermediate release rate.

The water soluble region of these macromers is preferably PEG. The watersoluble region can have multiple arms; for example, it may bestar-shaped or comb-shaped, as described, for example in U.S. Pat. No.5,410,016, incorporated herein by reference. The water soluble regionpreferably has 3, 4, 6, or 8 arms and a molecular weight of 500 to20,000, preferably, 1,000 to 10,000 daltons.

Methods for Increasing Protein Precipitation

The articles of the present invention, can be made to contain a BAS inan insoluble format, by combining the BAS with a molecule, or mixture ormolecules which preferentially excludes proteins, and a macromer,forming a mixture of these reagents, and polymerizing the mixture. Amolecule or mixture of molecules which preferentially exclude proteinscan be used in the formation of the article to increase proteinprecipitation. Examples of molecules which preferentially excludeproteins include, but are not limited to, macromers, poly(ethyleneglycol), hyaluronic acid, and poly(vinylpyrrolidone). A reagent whichcarries a positive or negative ion charge may be used in the formationof the articles of the invention in order to increase the precipitationof the BAS in the mixture which is then polymerized to form the article.The optimal reagent to be used depends on the charge of the protein,which is affected by the pH of the mixture. Examples of mixtures ofmolecules which preferentially exclude proteins include, but are notlimited to, a mixture of molecules comprising a positively chargedion-carrying reagent, for example, triethanolamine or Tris (for example,when the pH is such that the protein is negatively charged); or amixture of molecules comprising a negatively charged ion-carryingreagent, such as sodium dodecyl sulfate (for example, when the pH issuch that the protein is positively charged). A mixture comprising asurfactant, for example, Tween 20, Tween 80, or poloxamer F68, may alsobe used to increase the precipitation of the protein.

High Load and Low Burst Characteristics

A therapeutic agent, for example, a BAS may be readily incorporated inhigh yield into the articles described herein. For example, articles maybe prepared containing at least 5% active substance by dry weight.Preferably, the articles contain at least 10, 25, or 40% by dry weight.

As discussed above, the BAS of the present invention is in an insolubleformat when combined with a macromer and formed into an article. Thecombination of high load and the insoluble format of the activesubstance in the article provides the article with a slow releaseprofile, with little initial burst. These results are surprising giventhe view in the field of polymers that an article containing aninsoluble active substance will have large initial burst of the activesubstance.

The BAS contained in the articles of the present invention is insoluble.The formulation of articles containing an insoluble BAS may be achieved,for example, by mixing the BAS with PEG, and then combining thesereagents with the desired macromer.

The amount of BAS loaded into a microsphere may be measured by combiningit with a macromer and shaping into articles. The articles may then beplaced into an appropriate solvent, for example phosphate bufferedrelease media (0.01% NaN₃, 0.05 M PBS, pH 7.4) and assayed for theamount of BAS present by means available in the art, such asspectrophotometry.

Biologically Active Substances

A BAS that can be incorporated into the articles of the inventioninclude therapeutic, diagnostic, and prophylactic agents. They can benaturally occurring compounds, synthetic organic compounds, or inorganiccompounds. Substances that can be incorporated into the articles of theinvention include proteins, polypeptides, carbohydrates, inorganicmaterials, antibiotics, antineoplastic agents, local anesthetics,antiangiogenic agents, vasoactive agents, anticoagulants,immunomodulators, cytotoxic agents, antiviral agents, antibodies,neurotransmitters, psychoactive drugs, oligonucleotides, lipids, cells,tissues, tissue or cell aggregates, and combinations thereof.

Exemplary therapeutic agents include growth hormone, for example humangrowth hormone, calcitonin, granulocyte macrophage colony stimulatingfactor (GMCSF), ciliary neurotrophic factor, parathyroid hormone, andthe cystic fibrosis transmembrane regulator gene. Other specifictherapeutic agents include parathyroid hormone-related polypeptide,somatostatin, testosterone, progesterone, estradiol, nicotine, fentanyl,norethisterone, clonidine, scopolomine, salicylate, salmeterol,formeterol, albeterol, and valium.

Drugs for the treatment of pneumonia may be used, including pentamidineisethionate. Drugs for the treatment of pulmonary conditions, such asasthma, may be used, including albuterol sulfate, β-agonists,metaproterenol sulfate, beclomethasone dipropionate, triamcinoloneacetamide, budesonide acetonide, ipratropium bromide, flunisolide,cromolyn sodium, ergotamine tartrate, and protein or polypeptide drugssuch as TNF antagonists or interleukin antagonists.

Other therapeutic agents include cancer chemotherapeutic agents, such ascytokines, chemokines, lymphokines, and substantially purified nucleicacids, and vaccines, such as attenuated influenza virus. Substantiallypurified nucleic acids that can be incorporated include genomic nucleicacid sequences, cDNAs encoding proteins, expression vectors, antisensemolecules that bind to complementary nucleic acid sequences to inhibittranscription or translation, and ribozymes. For example, genes for thetreatment of diseases such as cystic fibrosis can be administered.Polysaccharides, such as heparin, can also be administered.

Other therapeutic agents include tissue plasminogen activator (t-PA),superoxide dismutase, catalase luteinizing hormone releasing hormone(LHRH) antagonists, IL-11 platelet factor, IL-4 receptor, enbrel, IL-1receptor antagonists, TNF receptor fusion proteins, megakaryocyte growthand development factor (MGDF), stemgen, anti-HER-2 and anti-VEGFhumanized monoclonal antibody, anti-Tac antibody, GLP-1 amylin, andGLP-1 amylin analogues.

Additional therapeutic agents include atrial natriuretic factor, atrialnatriuretic peptide, beta-human chorionic gonadotropin, basic fibroblastgrowth factor, bovine growth hormone, bone morphogenetic protein, B cellstimulating factor-1, B cell stimulating factor-2, bovine somatotropin,carcinobreaking factor, cartilage induction factor, corticotropinreleasing factor, colony stimulating factor, differentiating factor-1,endothelial cell growth factor, erythroid differentiation factor,elongation factor 1-alpha, epidermal growth factor, erythropoietin,thrombopoietin, thymopoietin, fibroblast growth factor, folliclestimulating hormone, granulocyte colony stimulating factor, glialfibrillary acidic protein, growth hormone releasing factor, humanalpha-1 antitrypsin, human atrial natriuretic factor, human chorionicgonadotropin, human leukemia inhibitory factor, hemopoietin-1,hepatocyte growth factor, human transforming growth factor, humanthyroid-stimulating hormone, interferon, immunoglobulin A,immunoglobulin D, immunoglobulin E, insulin-like growth factor-1,insulin-like growth factor-II, immunoglobulin G, immunoglobulin M,interleukin-1, interleukin-2, interleukin-3, interleukin-4,interleukin-5, interleukin-6, kidney plasminogen activator, lectin celladhesion molecule, luteinizing hormone, leukemia inhibitor factor,monoclonal antibody, macrophage activating factor, macrophage cytotoxicfactor, macrophage colony stimulating factor, megakaryocyte colonystimulating factor, tumor necrosis factor, macrophage inhibitory factor,Mullerian inhibiting substance, megakaryocyte stimulating factor,melanocyte stimulating factor, neutrophil chemotactic factor, nervegrowth factor, novel plasminogen activator, nonsteroidalanti-inflammatory drug, osteogenic factor extract, antitumor lymphokine,prostate-specific antigen, anti-platelet activating factor, plasminogenactivator inhibitor, platelet-derived growth factor, platelet-derivedwound healing formula, plasmatic human interleukin inducing protein,tumor angiogenesis factor, tissue control factor, T cell growth factor,T cell modulatory peptide, transforming growth factor, tumor growthinhibitor, tumor inhibiting factor, tissue inhibitor ofmetalloproteinases, tumor necrosis factor, tissue plasminogen activator,thyroid stimulating hormone, urokinase-plasminogen activator, vascularendothelial growth factor, and vasoactive intestinal peptide.

A preferred BAS is a substantially purified polypeptide or protein.Proteins are generally defined as consisting of 100 amino acid residuesor more; polypeptides are less than 100 amino acid residues. Unlessotherwise stated, the term protein, as used herein, refers to bothproteins and polypeptides. The proteins may be produced, for example, byisolation from natural sources or recombinantly. Examples includeinsulin and other hormones, including growth hormones, such as humangrowth hormone and bovine growth hormone. Other exemplary proteinsinclude Factor VIII, Factor IX, Factor VIIa, and anti-inflammatoryagents, such as interleukins, including interleukin-4. Other exemplaryproteins include enzymes, such as DNase and proteases. Other proteinsinclude cytokines, interferons, including interferon alpha andinterferon beta, poetins, angiogenic factors, differentiation factors,colony-stimulating factors, growth factors, ceredase, gibberellins,auxins, and vitamins, and fragments thereof. Exemplary growth factorsinclude vascular endothelial growth factor (VEGF), endothelial cellgrowth factor (ECGF), basic fibroblast growth factor (bFGF), andplatelet derived growth factor (PDGF).

Proteins are stable in the hydrogels of the present invention. Forexample, many of the proteins are protected from dimerization oraggregation, as discussed below in the Examples. The enzymaticdegradation of proteins or polypeptides can be further minimized byco-incorporating peptidase-inhibitors.

Treatment of an Animal Using Slow Release Protein Polymers

The polymer articles of the present invention may be used to treat ananimal, for example, a mouse, rat, or human, by delivering a BAS to theanimal. The articles may contain such a BAS as any of those describedabove. Various routes of administration may be used to deliver thearticles of the present invention, as described below.

The results of the treatment of an animal with therapeutic articlescontaining a BAS, as described herein, will vary according to the BASbeing delivered. For example, if hGH is delivered through thetherapeutic articles of the present invention, one would expect toobserve an increase in growth as a result of such a treatment. Iferythropoietin is delivered through the therapeutic articles, one wouldexpect to observe an increase in reticulocytes in the animal as a resultof the treatment. If insulin is delivered through the therapeuticarticles, then the treatment should result in a decrease in bloodglucose levels.

The articles of the present invention provide optimal delivery of a BAS,because they release the BAS in a controlled manner with a low bursteffect. The result of such a delivery rate is that the drug is deliveredsteadily over a desired period of time. A slower and steadier rate ofdelivery may in turn result in a reduction in the frequency with whichthe BAS must be administered to the animal. In addition, a low bursteffect may be highly desirable in some circumstances where the deliveryof too much BAS to a site is deleterious to the animal.

Routes of Administration of the Articles

Inhalation

The use of the hydrogel particles of the invention can enhance thedelivery of drugs to the lung. Administration to the lung provides forthe delivery of drugs that can be transported across the lung tissuebarriers and into circulation, as described WO 99/03454.

A problem with the delivery of active substances to the lung is thatpulmonary macrophages can take up the materials, thus preventing thematerial from entering into systemic and local circulation. Uptakeoccurs when proteins adsorbed to the particles' surfaces bind withreceptors on the surfaces of the macrophages. To prevent uptake, theinvention provides nonionic hydrogels, e.g., formed with polymers basedon polyethylene glycol. These hydrogels adsorb low levels of proteinsand thus bind poorly to cell surfaces. Anionic hydrogels, e.g., formedwith polyacrylic acid, also adsorb relatively low levels of proteins andthus bind poorly to cell surfaces.

In a further embodiment, biocompatible microcapsules may be formed andthe surface provided with water soluble non-ionic polymers such aspolyethylene oxide (PEO), to create resistance to cell adhesion, asdescribed in U.S. Pat. No. 5,380,536, hereby incorporated by reference.

The size and density of the particles can also be selected to maximizethe quantity of BAS that is delivered to the lung. For example, themacrophages will not take up large particles as efficiently as they willtake up small particles. However, large particles are not delivered tothe deep lung as well as small particles are. To overcome theseconflicting factors, the invention provides small particles that canswell as they hydrate. The particles are administered to the deep lungas small (i.e., 1-5 microns), dry, or slightly wet, particles; uponhydration, they swell, and therefore become resistant to uptake by thepulmonary macrophages. The swelling can occur when the particles arehydrated from the dry state and when they are hydrated from one state ofhydration to another by a change in temperature, pH, salt concentration,or the presence of other solvents, for example, depending upon thechemical and physical nature of the hydrogel polymer.

As used herein, the term “dry” means that the particles of the powderhave a moisture content such that the powder is readily dispersible inan inhalation device to form an aerosol. Preferably, the moisturecontent of the particles is below 10% by weight water, more preferablybelow about 5%, or optionally below about 2%, or lower.

The density of the particles is expressed in terms of tap density. Tapdensity is a standard measure of the envelope mass density. The envelopemass density of an isotropic particle is defined as the mass of theparticle divided by the minimum sphere envelope volume within which itcan be enclosed. The density of particles can be measured using a GeoPyc(Micrometers Instrument Corp., Norcross, Ga.) or a AutoTap (QuantachromeCorp., Boyton Beach, Fla.).

For example, the density of 3.4kL5 particles was determined as follows.3.4kL5 (1.0025 g), 200 mM TEOA in PBS; pH 7 (1.0260 g), and 1000 ppmEosin (0.1028 g) were combined. 200 mg of this solution was mixed withtalc (0.1015 g). The resulting suspension was placed in a 100 μl glasspipet and polymerized by light for 15 seconds (ILC Technology, Inc.Xenon Light Source with Fiber Optics). The rod was pushed out, placed onaluminum foil, and further polymerized for 3.5 minutes. The hardened rodwas lyophilized (vacuum 15E-3 mbar, trap temp. −50° C.) for 18 hours.The dry rod (water content<10%) was cut into small pieces, placed inheptane, and minced using a homogenizer (Silverson L4RT-A) at 5,000 rpmto small particles. The wet particles were air-dried, followed bynitrogen gas flow. The particles sizes ranged from 1 micron to 0.5 mm.

1.645 g of these particles was placed in a 10 mL graduated cylinder. Thegraduated cylinder was mounted on top of an Autotap densimeter(Quantachrome). The sample was tapped 100 times and the particles'volume was read. The process was repeated until no change in volume wasobserved. The final volume was 2.8 ml. The tap density of the particleswas 1.6435 g/2.8 ml=0.5870 g/ml.

In addition to particles, the polymer may be provided in other shapessuitable for delivery to the deep lung. For example, PEG emulsionmicrospheres are subjected to high pressure and a vacuum onto a flatplate to form very light very thin layers, for example, having a snowflake consistency, that react differently to fluidic wind forces. Theresulting thin flakes can be, e.g., 0.01 micron, 1 micron, or 10 micronsthick.

The particles can be administered to the respiratory system alone, or inany appropriate pharmaceutically acceptable excipient, such as a liquid,for example, saline, or a powder. Aerosol dosages, formulations anddelivery systems may be selected for a particular therapeuticapplication, as described, for example, in Gonda (“Aerosols for deliveryof therapeutic and diagnostic agents to the respiratory tract,” inCritical Reviews in Therapeutic Drug Carrier Systems, 6:273-313, 1990);and in Moren (“Aerosol dosage forms and formulations,” in: Aerosols inMedicine. Principles, Diagnosis and Therapy, Moren, et al., Eds.,Elsevier, Amsterdam, 1985).

Pulmonary drug delivery may be achieved using devices such as liquidnebulizers, aerosol-based metered dose inhalers, and dry powderdispersion devices. For the use of dry powder dispersion devices, thepolymer particle incorporating the therapeutic agent is formulated as adry powder, for example, by lyophilization or spray-drying. Methods forpreparing spray-dried, pharmaceutical-based dry powders including apharmaceutically acceptable amount of a therapeutic agent and a carrierare described in PCT WO 96/32149, hereby incorporated by reference.

Examples of a BAS that can be administered to the lung include, withoutlimitation, insulin, antitrypsin, calcitonin, alpha interferon, betainterferon, GLP-1, and DNAse.

Nasal Delivery

The articles of the present invention can also be used to administercompounds nasally. For example, a vaccine containing freeze dried orreconstituted microspheres can be administered nasally.

Intramuscular and Subcutaneous Administration

The articles of the present invention can be used to administermicrospheres that degrade over several days to 3 months, byintramuscular injection or by subcutaneous injection.

For example, growth hormone can be administered subcutaneously; thehormone leaves the microspheres at the site of injection as theydegrade. Growth hormone enters the systemic circulation, where, in turn,it exerts its effects directly, and indirectly through induction ofsomatomedin production in the liver and in other tissues. For thisapplication, particle sizes of up to 0.5 mm can be used.

In other embodiments, the active agent is a vaccine, such as tetanusvaccine, other proteins or polypeptides, or more complex immunogens. Thevaccine is released over time, from one week to many weeks, resulting inan improved immune response to the vaccine, compared to a bolusinjection followed by one or more booster shots with the same total doseof immunogen. Mixtures of different types of microspheres can result ininitial and booster shot-type immunization as well.

Intravenous Administration

Articles that contain a BAS useful in treating clotting disorders, suchas Factor VIII or Factor IX for hemophilia, can be administered byintravenous injection. The BAS is released over days to weeks. Atherapeutic level of the BAS is maintained that results in a betterclinical outcome. In addition, potentially lower total doses of a BAScan be administered, with a corresponding economic benefit. Theseapproaches help promote patient compliance.

In the case of intravenous injection, it is important to formulate themicrospheres in acceptable agents so the microspheres do not aggregateand clog blood vessels. The microspheres must be appropriately sized, sothat they don't lodge in capillaries. For this application, particlesizes of 0.2-0.5 microns are preferred.

In a number of inflammatory conditions, as part of the inflammatoryprocess that is mediated by selectin and ICAM expression/binding withneutrophil intravisation, blood vessels become leaky at the site ofinflammation. Hydrogel microspheres may be administered; thesemicrospheres will leak out of blood vessels at the site of inflammation,and then release their BAS payload locally over a period of time.Disease conditions where this approach may be useful could include, butare not limited to, inflammatory bowel diseases, asthma, rheumatoidarthritis, osteoarthritis, emphysema, and cystic fibrosis (with DNase asthe enzymatic drug).

Hydrogel microspheres that contain cytokines, lymphokines, or othercompounds to treat cancer can be administered by intravenous injection.Blood vessels within large solid tumors are generally leaky, and theblood flow within them is often slow. Thus, microspheres could lodgewithin solid tumors and release their anticancer BAS locally, eitherkilling tumor cells directly or by activating the immune system locally.This approach could be used, for example, with compounds such asinterleukin 2, where the systemic and local toxicity has been doselimiting and where the resulting side effects are significant.

The microspheres of the present invention may be cleared relativelyslowly from the circulation. Alternatively, the microspheres can betargeted to exit the circulatory system through leaky blood vessels orthrough more active targeting mechanisms, e.g., receptor mediatedtargeting mechanisms.

Oral Administration

In some portions of the gastrointestinal tract, there is relatively goodtransport of proteins across the intestinal mucosa into the systemic andlocal circulation. The compositions of the invention, for example,freeze dried microspheres containing protein (with very small particlesizes), can therefore be administered orally in an appropriate entericformulation that protects the drug-containing microspheres fromenzymatic attack and the low pH found in the upper GI tract. Such anenteric formulation could also be designed using several availabletechnologies to gradually expel BAS-containing microspheres as theenteric capsule traverses the gastrointestinal tract. This is describedin more detail in WO 99/03454 and in Mathiowitz et al. (Nature 386:410-414 (1997)). It is anticipated that this approach will have a numberof advantages over other approaches for delivering proteins and othermolecules, even small molecules, orally. First, PEG and proteins arecompatible, so the major manufacturing and stability problems found withother drug delivery approaches can be avoided. Secondly, dried hydrogelsare very adhesive to wet tissue. The microparticles will bind well tothe GI tract and will be transported into the system via thegastrointestinal circulation or release their contents on the intestinalmucosa; in turn, the drug will enter the systemic and gastrointestinalcirculation. Chemical enhancers, or formulations containing compositionsthat utilize specific and non-specific biological transport mechanismsto facilitate transport across the GI tract into the systemiccirculation, can be included as well.

Targeting

Targeting ligands can be attached to the particles via reactivefunctional groups on the particles. Targeting ligands permit bindinginteractions of the particle with specific receptor sites, such as thosewithin the lungs or those on endothelial cells specific to differentregions in the body's microvasculature. A targeting ligand is selectedwhich specifically or non-specifically binds to particular targets.Exemplary targeting ligands include antibodies and fragments thereofincluding antibody variable regions, lectins, hormones, or other organicmolecules capable of specific binding to receptors on the surfaces ofthe target cells. Other ligands are described in Science (279:323-324(1998)), hereby incorporated by reference.

Microspheres can be made with both a BAS and a targeting molecule.Double microspheres can also be made, in which the inner sphere containsdrug and the outer PEG shell contains the targeting molecule or reagent.

Excipients and Carriers

The particles incorporating a therapeutic agent or diagnostic agent maybe provided in combination with one or more pharmaceutically acceptableexcipients available in the art, as described, for example, in PCT WO95/31479, hereby incorporated by reference. Excipients may be selectedthat can, in some applications, enhance stability, dispersability,consistency, and bulking to ensure uniform pulmonary delivery. Theexcipient may be, e.g., human serum albumin (HSA), bulking agents suchas carbohydrates, amino acids, polypeptides, pH adjusters or buffers,and salts. Additional excipients include zinc, ascorbic acid, mannitol,sucrose, trehalose, cyclodextrans, polyethylene glycol, and othercommonly used pharmaceutical excipients, including those described inThe United States Pharmacopeia, published by the United StatesPharmacopeia Convention, Inc., 1995 (see, e.g., pp. 2205-2207).Exemplary carbohydrates include monosaccharides, such as galactose, anddisaccharides such as lactose. Excipients that stabilize proteins areespecially useful.

In some cases, the excipients are used as carriers; i.e., they are usedto modulate the release rate of the active substances. For example,mannitol can be used to accelerate or delay release.

There now follow particular examples that describe the preparation ofcompositions of the invention, and the methods of the invention. Theseexamples are provided for the purpose of illustrating the invention, andshould not be construed as limiting.

In some of the following Examples a macromer made of a triad ABA blockcopolymer of acrylate-PLA-PEG-PLA-acrylate was used. The PEG had a MW of3,400 daltons; the poly(lactic acids) on both sides had an average ofabout five lactate units per side; they are therefore referred to hereinas “3.4kL5.” When a lower molecular weight PEG, such as 2,000 daltonswas used, the resulting macromer is abbreviated as “2kL5.”

In other Examples an acrylate-PCL-PEG-PCL-acrylate macromer was used.The PEG had a MW of 3,400 daltons and had polycaprolactone on bothsides, with an average of about 6 caproyl units per side. The polymer isreferred to herein as “3.4kC6.”

In yet other Examples a 3-arm macromer was used. This macromer consistedof a PEG core with 3 arms, 3 lactate groups attached to each arm of thePEG. The PEG had a MW of 4,200 daltons and the polymer is referred toherein as “4.2kL3-A3.”

EXAMPLE 1

General Preparation of a Macromer Solution

The protein was weighed out, and the following components were added tothe protein: (i) 90 mM TEOA/PBS, pH 8.0; (ii) 35% n-vinyl pyrrolidinone(n-VP); and (iii) 1000 ppm Eosin. The resulting mixture was stirred wellusing a spatula. The solution was kept in the dark for about 10 minutes,or until the macromer had absorbed all of the solution, or until thesolution was homogenous.

Macromer solutions having the following ingredients were prepared.

Amount Amount Amount Amount 90 mM 35% 1000 ppm Amount Amount TotalProtein TEOA n-VP Eosin 3.4kL5 2kL5 amount 15 mg 57 mg 15 mg 3 mg 45 mg 0 mg 135 mg 15 mg 57 mg 15 mg 3 mg  0 mg 45 mg 135 mg

EXAMPLE 2

Precipitation of hGH and Formulating into Hydrogel Microspheres

To a 100 mg/ml hGH solution in 5 mM ammonium hydrogen carbonate buffer77 μl of a 1300 mM triethanolamine, pH 8, solution was added. Upon theaddition of 400 mg of PEG 2K to the above solution, a fine precipitateof hGH was formed. The sample was centrifuged at 4000 rpm for severalminutes and 0.9 ml of the supernatant was removed. To the precipitatedmixture, 1 g of 4.2kL5-A3 macromer was added, followed by the additionof 0.1 ml of a 10 mM Eosin Y solution. The mixture was then emulsifiedin an oil phase to form microspheres which were polymerized using anargon laser. The in vitro release characteristics of this formulationare shown in FIG. 1. No burst was observed and release continued for atleast 5 days.

EXAMPLE 3

Micronization of Freeze-Dried Human Growth Hormone and Formulation intoHydrogel Microspheres

A 10 mg/ml solution of hGH ammonium acetate was frozen and freeze-dried,resulting in a dry cake of pure hGH. The following macromer solution wasprepared: 1 g 2kL5, 1.2 g phosphate buffered saline (pH 7), 0.4 g of asolution of 25% trehalose and 0.4% F-68 in water, 0.24 g of a 10%solution of 2,2-dimethoxy 2 phenyl-acetophenone in tetrahydrofuran. Tothis solution was added 0.2 g of the freeze-dried hGH. Following mixingto disperse the hGH, the hGH in suspension was further micronized by 20passages through a 1.5 inch 20 g needle. This suspension was thenemulsified in an oil phase to form microspheres which were polymerizedby exposure to UV light (365 nm) for 3 min. The in vitro releasecharacteristics of this micropheres are shown in FIG. 2.

EXAMPLE 4

Formation of Articles

The microspheres, in the form of a hydrogel, were placed onto asilanized glass slide. Using pieces of plastic sheets with thicknessesof about 0.4±0.2 mm as spacers, another silanized glass slide was placedon top and held firmly in place using binder clips.

A light source (ILC Technology, Inc. Xenon Light Source with FiberOptics) was adjusted to about a 5-cm distance. The center of the diskwas illuminated; both sides of the disk were illuminated for two minuteseach, to form an opaque disk.

EXAMPLE 5

Preparation of Articles Containing 4.2kL3-A3 Macromers and bSA

To 1 ml of 50% ethanol/water solvent, 1 g of 4.2kL3-A3 macromer wasadded with 7.8 mg of 2,2-dimethoxy-2-phenyl-acetophenone (DMPA). Oncomplete dissolution of the macromer and DMPA, 250 mg of spray dried bSAwas added and stirred until the mixture was uniform. The entire mixturewas then emulsified in 200 g of a 0.5% lecithin in heavy white mineraloil solution stirred at 600 rpm. Shortly afterwards, the disperseddroplets were photo-polymerized using a Black-Ray B100AP UV lamp for aperiod of 15 minutes. The in vitro release profile of this formulation,shown in FIG. 3. indicates no burst.

A degradable macromer (4.2kL3-A3) was combined with bSA. The protein wasloaded at a loading of 20%, based on dry weight. An emulsion was formedusing white heavy mineral oil. Polymerization of the macromer into ahydrogel then occurred through spray drying and UV polymerizationtechniques.

EXAMPLE 6

Analyses of Biological Active Substance Release from a Macromer

After formation of the articles, as described, for example, in Example4, the disks were removed and weighed on a clean, tared silanized glassslide. The disk was placed into a heat-sealed membrane bag, as describedin more detail below. One 20 μl disk was placed in each bag. The bag washeat-sealed, placed in 2.0 ml of phosphate buffer release media (0.01%NaN₃, 0.05 M PBS; pH 7.4), placed on an orbital shaker turning at 100rpm, and incubated at 39° C.

For each time point, the bag was placed into fresh 2.0 ml of PBS ReleaseMedia. Samples were collected for analysis every day for as long as theBAS was being released.

Membrane bags were prepared as follows. Membrane sheets were cut intopieces of approximately 7×2.5 cm. The sheets were folded in half. Usinga Bunsen burner or a propane torch, a spatula was heated until it becamered. The edges of the sheets were aligned, and the side of the membranewas cut with the red-hot tweezer to seal the sides. Once the disk wasplaced into the bag, the last side was sealed using the sameheat-sealing technique.

The samples were analyzed daily by SEC-HPLC. Monomers, dimers, andsoluble aggregates could be detected using this method. The mobile phaseused was 0.08 M TFA in 60/40% CH₃CN/H₂0, adjusted to pH 2.0, isocratic,with a flow rate of 1.5 ml/min. The signals were detection at awavelength of 220 nm. The column used was a Bio-Rad Bio-Sil® SEC 250, 5micron particle size, 300×7.8 mm ID, equipped with a guard column(Bio-Rad Bio-Sil® R SEC 250 Guard, 5 micron particle size, 80×7.8 mmID). The injection volume was 10 μl. The standard calibration curveswere 0, 0.1, 0.25, 0.5, 0.75, and 1 mg/ml bST in the mobile phase.

EXAMPLE 7

Production of Microspheres with Efficient Protein Loading and Low BurstEffects

FIG. 3 shows an example of the high protein loading and low burstcharacteristics of the therapeutic articles of the present invention.The articles contain 4.2kL3-A3 macromers combined with bSA (in either amonomer or dimer form) and formed into microspheres using spray dryingtechniques. The bSA was loaded at a calculated loading of 20%. Releaseof bSA from the microspheres was assayed as described above. The releaseoccurred at a slow steady rate, and no burst effect was exhibited. Aftera period of 9 days, less than 30% of the bSA was released from macromerscontaining bSA. These results demonstrate that the therapeutic articlesof the present invention can provide slow release of a BAS, with littleor no burst effect.

EXAMPLE 8

Effect of the Particle Size of a BAS on Release of bSA from 30% 3.4kL5

The effect of the particle size of a BAS on its release from an article,for example, a microsphere was also determined. The bSA used to form themicrospheres was either ground under liquid nitrogen into fine particlesof less than approximately 75 microns, or were left unground.Microspheres containing 30% 3.4kL5 and either the fine-ground orunground bSA were formed using the methods described above, and wereassayed for the release of bSA, loaded at 25%, based on dry weight. FIG.4 illustrates the results of these studies. Compared to the microspherescontaining unground bSA, the microspheres containing fine-ground bSAreleased its bSA over a longer period of time. In addition, themicrospheres containing the fine-ground bSA exhibited steady rate ofrelease (releasing less than 20% of the total bSA loaded within thefirst 24 hours), with no burst effect, while the microspheres containingthe unground bSA exhibited a burst effect (releasing approximately 50%of the total bSA loaded within 24 hours). These results demonstrate thatmicrospheres containing a BAS which has a small particle size provideslower release profiles and low burst effects.

EXAMPLE 9

Effects of BAS Particle Size and Protein Loading on the Release ofFine-Ground bSA (10% Loaded) and Various Crystalline Particles (1-10%Loaded) from 3.4kL5

The effects of the particle size of a BAS and protein loading on therelease of bSA from an article, was also examined (FIG. 5). The bSA usedto form the microspheres was either ground under liquid nitrogen intofine particles of less than approximately 75 microns, or were leftunground. The fine-ground bSA was combined with 3.4kL5 to form 30%3.4kL5 microspheres loaded with 10% bSA, using the methods describedabove. The unground bSA, containing various particle sizes was combinedwith 3.4kL5 to form 30% 3.4kL5 microspheres loaded with 1-10% bSA. Themicrospheres were then assayed for the release of bSA. FIG. 5illustrates the results of these studies. Compared to the microspherescontaining unground bSA, the microspheres containing fine-ground bSA,loaded at 10%, released its bSA over a longer period of time. Inaddition, the microspheres containing the fine-ground bSA exhibited alower burst effect (releasing approximately 20% of the total bSA loadedwithin the first 24 hours) than its unground counterpart (releasingalmost 50% of the total bSA loaded within the first 24 hours). Theseresults demonstrate that microspheres containing a BAS which has a smallparticle size and is highly loaded provide a desirable BAS releaseprofile compared to microspheres containing various particle sizes and alower load of BAS.

EXAMPLE 10

Effect of Protein Particle Size on Release of hGH from 30% 3.4kL5

The effect of the particle size of a BAS on its release frommicrospheres was further examined using microspheres containing eitherfine-ground or unground hGH. The hGH used to form the microspheres waseither ground under liquid nitrogen into fine particles of less thanapproximately 75 microns, or were left unground. Microspheres containing30% 3.4kL5 and either the fine-ground or unground hGH were formed usingthe methods described above, and were assayed for the release of hGH,loaded at 25%, based on dry weight, and 10% as manufacturing conditions.FIG. 6 illustrates the results of these studies. Compared to themicrospheres containing unground hGH, the microspheres containingfine-ground hGH released its hGH over a longer period of time. Inaddition, the microspheres containing the fine-ground hGH exhibited asteady rate of release (releasing less than 40% of the total hGH loadedwithin the first 24 hours), with no burst effect, while the microspherescontaining the unground hGH exhibited a high burst effect (releasingapproximately 70% of the total hGH loaded within 24 hours). Theseresults demonstrate that microspheres containing a BAS which has a smallparticle size provide the characteristics which are highly suitable fordelivery of agents for therapeutic use: slow protein release and lowburst effects.

EXAMPLE 11

Effect of Microsphere Pore Size on Release of hGH from Macromers

The effect of the pore size of the microspheres containing fine-groundhGH on the release rate of the hGH was also examined (FIG. 7).Microspheres containing hGH, loaded at 25%, based on dry weight, and 10%as manufacturing conditions, and either 30% 2kL5 or 30% 3.4kL5 wereformed using the techniques described above. The microspheres containing2kL5 had a smaller pore size than the microspheres containing 3.4kL5.The release rate of hGH from these microspheres was then assessed usingtechniques described above. These studies showed that fine-ground hGHwas release from 2kL5-containing microspheres at a slower rate than itwas released from 3.4kL5-containing microspheres. These results suggestthat macromers which result in a smaller microsphere pore size release aBAS at a slower rate than those which result in a larger microspherepore size.

EXAMPLE 12

Controlled Release of Bovine Somatotropin in Hypophysectomized Rats

The controlled delivery of active bovine somatotropin (MW 20 Kd) wasconfirmed in the hypophysectomized rat model. Hypophysectomized femalerats were purchased from Taconic Labs (Germantown, N.Y.). The rats wereweighed each morning. Prior to the initiation of the study, the ratswere held 7 days to confirm a lack of significant growth. On day 1 ofthe study the rats were weighed. The rats were then divided into 3groups of equal mean weights. Group 1 remained untreated and served as anegative control. Group 2 received an implant of bST in a hydrogel madeof a blend of 3:1 of 3.4KL5 and poly(ethylene glycol) diacrylate (eachdevice contained 0.9 to 1.1 mg of bST). The rats in Group 3 wereinjected with 100 μg bST subcutaneously each day for the duration of thestudy.

At the end of the 12 day treatment period, the rats were analyzed fortheir growth over the period of treatment. The rats of Groups 1 did notgrow significantly, while the rats of Groups 2 and 3 grew at ratesfaster than Group 1 and approximately equal to one another.

EXAMPLE 13

Controlled Release of Erythropoietin in Rats

The controlled delivery of active human erythropoietin (EPO) wasconfirmed in male Sprague-Dawley rats purchased from Taconic Labs(Germantown, N.Y.). Hydrogel devices were manufactured to contain 3000Units of EPO per device. One of these devices was implanted in each of anumber of rats (Group 1). Another group of an equal number of rats(Group 2) received a subcutaneous injection of EPO (1000 Units) dailyfor 3 days. A third group of rats (Group 3) received no treatment.

On day 5 after implantation of the device and the start of thesubcutaneous injections, venous blood samples were obtained from eachrat and stored in EDTA. The fraction of reticulocytes (immature redblood cells) was determined after staining with Acridine Orange byautomated flow cytometry. The rats in Group 1 had 18% reticulocytes, therats of Group 2 had 15% reticulocytes, and the rats in Group 3 had 4%reticulocytes.

EXAMPLE 14

Controlled Release of Insulin in Diabetic Rats

Sprague-Dawley rats were purchased from Taconic Labs (Germantown, N.Y.).Diabetes was induced by treatment with streptozotocin (65 mg/kg, i.v.)and confirmed 48 hours later by elevation of blood glucose (>300 mg/dl).Following anesthetization of the rat with pentobarbital (35 mg/kg), acatheter was placed in a jugular vein. After a baseline blood sample wastaken for the determination of blood glucose concentration, a hydrogeldevice containing 1 Unit of insulin was implanted subcutaneously. Bloodsamples were taken at 15, 30, 60, 120, and 180 minutes afterimplantation of the device and used to determine blood glucose levels.

The blood glucose level of the rat implanted with the hydrogel devicedecreased, demonstrating that the devices was capable of releasinginsulin in its active form.

To test the pulmonary delivery system for insulin-containing hydrogelparticles, the neck of the rat was opened with a midline incision andthe trachea was exposed by blunt dissection. A slit was cut into thetrachea, and a small polyethylene tube was advanced distally into thelung. A small volume of insulin-containing hydrogel microparticles(total dose of 3 Units of insulin) was instilled into the lung and thetube was removed. Blood samples were taken and analyzed as describedabove for the subcutaneous device.

The blood glucose levels dropped significantly within 30 minutes andremained low (below 150 mg/dl) for at least 180 minutes.

EXAMPLE 15

Controlled Release of Human Growth Hormone in Hypophysectomized Rats

The controlled delivery of active human growth hormone (hGH, MW 20 Kd)is confirmed in the hypophysectomized rat model. Hypophysectomizedfemale rats purchased from Taconic Labs (Germantown, N.Y.) are weighedeach morning. Prior to the initiation of the study the rats are held 7days to confirm lack of growth. The rats are divided into 3 groups ofequal mean weights. Group 1 remains untreated and serves as a negativecontrol. Group 2 receives an implant of hGH in a hydrogel made of a 3:1blend of 3.4kL5 and 3.4kC6 (each device containing approximately 1 mg ofhGH). The rats in Group 3 are injected with 100 μg hGH subcutaneouslyeach day for the duration of the study.

It is expected that the untreated control group will not grow during thestudy, and that the rats of Groups 2 and 3, receiving the hGH hydrogelimplant and 100 μg hGH injections daily during the study, respectively,will exhibit continued growth.

EXAMPLE 16

Pulmonary Devices Containing Human Growth Hormone (hGH)

To a 20 ml vial are added: 0.2559 g of 200 mM of TEOA (in PBS buffer; pH7.0), 0.2548 g of 3.4KL5, 0.0206 g of 1000 PPM eosin (in PBS; pH 7.0),and 0.0615 g hGH (Genentech's hGH injectable formulation, purified by aMillipore Centricon™). The resulting mixture is stirred and placed into10 ml glass tubes. The tubes are exposed to xenon light (ILC Technology,Inc. Xenon Light Source with Fiber Optics) for 10 seconds. Thesemi-cured hydrogel is pushed out of the glass tube and furtherpolymerized for 3.5 minutes. The cured hydrogel rods are put into 15 mlof heptane and are ground using a homogenizer (Silverson L4RT-A) for 30seconds at 5000 rpm, followed by 30 seconds at 3000 rpm. The heptane isdecanted, and the powder is dried under nitrogen. The powder is used forpulmonary, oral, or subcutaneous sustained delivery of hGH.

EXAMPLE 17

Oral Formulation for Release of Proteins

Using the techniques described above, insulin, human growth hormone,human alpha interferon, or erythropoietin is incorporated into macromerparticles. Using cryomilling or other milling procedures known in theart, very small microparticles are produced, preferably of an averagesize of less than about 500 nanometers. Such nanoparticles are thenintroduced into the rat GI tract surgically, using catheter infusioninto the upper GI tract. The dosing of such nanoparticles is based uponthe assumption that about 0.5% of the drug in the nanoparticles will bedetectable in the blood of such rats, e.g., by RIA, with the specificpharmacology of each drug taken into account.

In the case of insulin, blood samples are taken at time t=−15, 0, 30,60, 90, 120, and 180 minutes, and monitored for insulin by RIA and forblood glucose by glucometer (when insulin is being administered,diabetic rats are utilized).

For other drugs, normal rats are used and blood drug levels are measuredat these same time points using RIA or ELISA techniques.

In addition to the above procedures, the above drug-containingmicrospheres can be modified to enhance their absorption in the smallintestine, colon, and other appropriate areas of the GI tract. Suchmodifications can include precipitating lipid bilayers around themicrocapsules so they appear as fat-like particles from digested food,linking molecules such as ferritin to the particles, or putting acharged layer on the outside of the microparticles.

EXAMPLE 18

Evaluation by Reverse Phase HPLC

Microspheres were prepared by first adding 0.154 ml of 3Mtriethanolamine solution at pH 8.0 (TEOA) to approximately 2 ml of 100mg/ml solution of hGH in ammonium bicarbonate and then mixing well.Next, about 800 mg of solid PEG 2k was added and mixed with a spatula,resulting in a very small amount of precipitated hGH in the solution.Samples were then centrifuged at 4000 rpm for thirty minutes and about1.8 g of supernatant was removed. About 1 g of macromer (4.4k PEGtris(lactate)₃ triacrylate) was added to each centrifuge tube andstirred. Next, about 0.1 to 0.15 mL of TEOA was added, followed by 0.05mL of 40 mM Eosin Y. The samples were then centrifuged for three minutesat 4000 rpm. Samples were then polymerized by forming a disc uponexposure to light as described in Example 4 or by first making amicroemulsion by mixing with oil (PPG 2k) and then exposing to light asdescribed in Example 4.

The resulting microspheres were analyzed by Reverse Phase HPLC(RP-HPLC). Samples were prepared for RP-HPLC by first extracting hGHfrom the microspheres with NaOH. Briefly, 10 mg of microspheres wasadded to 1 mL of NaOH(1N)/Tris (50 mM, pH 7.5) (1:9, v/v) solution andincubated at ambient temperature for 5 minutes. 5N HCl was used totitrate the solution to a final pH of 7.5. The sample was thenmicrocentrifuged for 2 minutes and filtered through a 0.45 micronfilter. 100 μL of the hGH solution was injected onto a Vydac C-4 column(214TP54) equilibrated and run under isocratic conditions at 0.5 mL/min.employing n-propanol/Tris (50 mM, pH 7.5)(29:71, v/v) as a solvent.Separation was performed at column temperature of 45° C. over 50 minuteswith UV detection at 220 nm. hGH eluted at a retention time of 33±3minutes. Results are shown in Table I. The term “% RP” refers to thepercentage of protein (hGH) that is not found in the monomer peak, whichmay include forms of hGH that are normally found in commerciallymarketed hGH preparations and that are active and safe, such as oxidizedand deamidated forms.

TABLE I Sample No. % RP  18-86-1 41 318-1 48 318-2 43

Other batches of microspheres were prepared by first adding 0.154 ml of3M Tris buffer at pH 6.0 to about 2 ml of 100 mg/ml hGH solution inammonium bicarbonate and mixing well. To this solution, about 800 mg ofPEG 10k was added as solid to obtain precipitated protein. Samples werethen centrifuged at 4000 rpm for thirty minutes and about 1.8 g ofsupernatant was removed. About 1 g of macromer (4.4k PEG tris(lactate)₃triacrylate) was added to each centrifuge tube and stirred. Next, about0.1 to 0.15 mL of TEOA was added, followed by 0.05 mL of 40 mM Eosin Y.The samples were then centrifuged for three minutes at 4000 rpm. Sampleswere then polymerized by forming a disc upon exposure to light asdescribed in Example 4 or by first making a microemulsion by mixing withoil (PPG 2k) and then exposing to light as described in Example 4. Onesample, Sample No. 27-50, was prepared using PEG 20k.

The resulting microspheres were analyzed by Reverse Phase HPLC. Sampleswere prepared for RP-HPLC either by the NaOH extraction method describedabove or by the following cryogrinding method. Results are shown inTable II, with the sample preparation method indicated. Approximately 10mg of microsphere sample was weighed in a microcentrifuge tube. A pestlewas placed in the tube and then the tube was immersed in a liquidnitrogen bath for approximately one minute. With the tube still in theliquid nitrogen bath, the sample was ground for approximately fiveminutes. The tube was then removed and allowed to stand forapproximately two minutes at ambient temperature. The ground sample wasthen suspended in about 1 mL of 25 mM potassium phosphate buffer, pH6.5. The pestle was removed, and the sample incubated for about tenminutes at ambient temperature. The sample was then centrifuged at 15000rpm for about five minutes to obtain a clear aqueous phase. Thesupernatant was the filtered through a 0.45 micron filter. RP-HPLC wasperformed by injecting 100 μL of sample onto a Vydac C-18 column(218TP54) equilibrated and run under isocratic conditions at 1 mL/min,using n-propanol/potassium phosphate (25 mM, pH 6.5) (27:73, v/v) as asolvent. Separation was performed at a column temperature of 55° C. over30 minutes with UV detection at 220 nm.

TABLE II Sample No. % RP 27-32 26 (NaOH) 27-50 19 (NaOH) (Tris, PEG 20k)320-4  20 (NaOH) 502 15.2 (NaOH) 507 17 (NaOH) 508 15 (NaOH) 530 16(NaOH) 548 10.4 (Cryogrind) 556 7.7 (Cryogrind) 557 8.7 (Cryogrind) 5618.5 (Cryogrind) 570 6.2 (Cryogrind) 575 7.2 (Cryogrind)

By comparison, microspheres made without a molecule that preferentiallyexcludes proteins (“Control” in Table III), yield % RP values of 53%using the NaOH extraction method, and 23% using the cryogrind method.Also presented in Table III is a comparison of the two samplepreparation methods.

TABLE III Sample No. % RP NaOH Method % RP Cryogrind Method Control 5323 361 30.2 15 362 28 15

Other Embodiments

From the foregoing description, it will be apparent that variations andmodifications may be made to the invention described herein to adopt itto various usages and conditions. Such embodiments are also within thescope of the following claims.

All publications and patents mentioned in this specification are hereinincorporated by reference to the same extent as if each individualpublication or patent was specifically and individually indicated to beincorporated by reference.

1. A biocompatible therapeutic article comprising, a macromer havingpolymerized end groups, precipitated human growth hormone, and amolecule or mixture of molecules which preferentially excludes proteins,wherein said molecule or mixture of molecules is present in an amountsufficient to reduce the solubility of said human growth hormone in saidarticle to less than 10 mg/ml.
 2. The biocompatible therapeutic articleof claim 1, wherein said molecule which preferentially excludes proteinsis selected from the group consisting of a macromer, poly(ethyleneglycol), hyaluronic acid, and poly(vinylpyrrolidone).
 3. Thebiocompatible therapeutic article of claim 1, wherein said macromercomprises: (a) a region forming a central core; (b) at least twodegradable regions attached to said core; and (c) at least twopolymerized end groups, wherein said polymerized end groups are attachedto said degradable regions.
 4. The biocompatible therapeutic article ofclaim 3, wherein said central core comprises a polymer selected from thegroup consisting of poly(ethylene glycol), poly(ethylene oxide),poly(vinyl alcohol), poly(vinylpyrrolidone), poly(ethyloxazoline),poly(ethylene oxide)-co-poly(propylene oxide) block copolymers,polysaccharides, carbohydrates, proteins, and combinations thereof. 5.The biocompatible therapeutic article of claim 3, wherein saiddegradable regions comprise a polymer selected from the group consistingof poly(α-hydroxy acids), poly(lactones), poly(amino acids),poly(anhydrides), poly(orthoesters), poly(orthocarbonates), andpoly(phosphoesters).
 6. The biocompatible therapeutic article of claim1, wherein said article comprises at least 5% human growth hormone bydry weight.
 7. The biocompatible therapeutic article of claim 1, whereinsaid article comprises at least 10% human growth hormone by dry weight.8. The biocompatible therapeutic article of claim 1, wherein saidmolecule or mixture of molecules is present in an amount sufficient toreduce the solubility of said human growth hormone in said article toless than 1 mg/ml.
 9. The biocompatible therapeutic article of claim 1,wherein the time at which 5% of the releasable human growth hormone isreleased from the article is greater than 1/16 of t₅₀.
 10. Thebiocompatible therapeutic article of claim 1, wherein said human growthhormone is released from said article such that t₅₀ is greater than orequal to ⅝ of t₈₀.
 11. The biocompatible therapeutic article of claim 1,wherein said articles release at least 80% of the human growth hormoneat a time 1¼ times eater than t₅₀.
 12. The biocompatible therapeuticarticle of claim 1, wherein said article has a particle size of lessthan about 75 microns.
 13. The biocompatible therapeutic article ofclaim 1, wherein said macromer has a water soluble region comprisingpoly(ethylene glycol) of about 500 to 20,000 daltons.
 14. Thebiocompatible therapeutic article of claim 1, wherein said mixture ofmolecules comprises a positively charged ion-carrying reagent.
 15. Thebiocompatible therapeutic article of claim 1, wherein said mixture ofmolecules comprises a negatively charged ion-carrying reagent.
 16. Thebiocompatible therapeutic article of claim 1, wherein said mixture ofmolecules comprises a surfactant.